Research indicates that the H5N1 avian influenza virus that infected human in Hong Kong in 1997 has 8 gene fragments, all derived from Eurasian avian influenza and has the capacity of binding the receptor of α(2,3) saliva acid, showing the typical characters of avian influenza viruses (Matrosovich, M. et al., J Virol, 1999, 73(2):1146-55). And the H5N1 avian influenza viruses of 1997 were cleared away after slaughtering all the poultry in Hong Kong. However, the virus A/goose/Guangdong/1/96(H5N1) from which the H5N1 in 1997 is derived was prevalent successively in the southeast of China (Cauthen, A. N. et al., J. Virol., 2000, 74(14):6592-9; Webster, R. G. et al., J. Virol., 2002, 76(1):118-26), and soon the virus was substituted by different kinds of genotypes (Guan, Y. et al., Proc Natl Acad Sci USA, 2002, 99(13):8950-5) which were lethal for chicken but not for duck. And these H5N1 viruses were cleared away again by slaughtering all the poultry until substituting viruses of new genotypes came up in 2002. Between 1997 and 2001, HA of these virus strains of different genotypes have similar immunogenicity. But obvious antigen drift occurred in 2002 (Guan, Y. et al., Proc Natl Acad Sci USA, 2004, 101(21): 8156-61; Sturm-Ramirez, K. M. et al., J. Virol., 2004, 78(9): 4892-901). It is rare that the typical character of the H5N1 virus of late 2002 is resulting in high mortality for duck and other waterfowl. Death of large number of waterfowl happened last time in 1961 when lots of terns died because of A/tern/South Africa/61(H5N3).
In early February of 2003, a family of Hong Kong was infected by H5N1 virus (Peiris, J S et al., Lancet, 2004, 363 (9409):617-9). The daughter of the family died of respiratory tract infection of unknown reason during her visit to Fujian, the father and the son had acute respiratory illness after returning to Hong Kong. Eventually the father died but the son recovered from the disease. Both the father and the son have been diagnosed as infected by the H5N1 influenza viruses that are highly similar in antigenecity and molecular level with the viruses which are highly lethal for chicken and duck after antigen drifting. (Guan, Y. et al., Proc Natl Acad Sci USA, 2004, 101 (21):8156-61).
Unprecedented large-scale avian influenza occurred in Asia in 2004 including the H5N1 appeared in China, Japan, Korea, Thailand, Vietnam, Indonesia, Cambodia and Laos, the H7N3 in Pakistan, and the H5N2 in Taiwan. A large number of poultry especially chicken were slaughtered because of the pandemic infection of avian influenza. Up to now, cases of human infection of avian influenza appeared in those countries where large-scale epidemic spread happened, and many of those infected died. These H5N1 viruses were first found in dead migratory waterfowl including egrets, gray herons and Canada geese in Hong Kong in November 2002. After analyzing the viruses obtained from the dead birds, it was found that comparing to the previous virus in Hong Kong, the antigenecity of these viruses has severely changed and these viruses have got the capacity of causing the death of large number of infected duck. (Sturm-Ramirez, K. M. et al., J. Virol., 2004, 78(9): 4892-901). The unusual aspect of this event is that all the high pathogenic H5N1 viruses were isolated from the migratory birds flying to Hong Kong in the winter of 2002-2003. And at that time some people predicted that the migratory birds will spread the virus after they fly back in summer (Sturm-Ramirez, K. M. et al., J. Virol., 2004, 78(9): 4892-901). This was true, large number of migratory birds were infected with H5N1 in Qinghai Lake, and according to research, all the present H5N1 viruses in Europe and Africa were brought from Asia by migratory birds.
Genetically, the pandemic H5N1 virus in Asia in 2004 was very similar to the Z-genotype of the virus that appeared in Hong Kong in 2003 and became the mainly spreading virus. At this time the Asia pandemic virus was very similar to A/Vietnam/1203/04(H5N1) in antigenic and genetic level and have all 8 gene fragments derived from Eurasian avian influenza. In both Vietnam and Thailand, cases of human infected by virus of this genotype appeared.
The pandemic H5N1 viruses have the potential of successfully transmitting to mammals. Because of successive evolution and the tendency of recombination, a variety of different genotypes of H5N1 viruses were produced, antigenic drift of the HA of H5N1 viruses occurred and the viruses have already had high pathogenic capacity for waterfowl.
The WHO (World Health Organization, WHO) statistics shows that currently Asia, Africa, Europe and North America all have reported poultry infected with highly pathogenic avian influenza virus [Organization, WH, H5N1 avian influenza: Timeline of major events. 2007]. From 2003 to Apr. 2, 2007, in 12 countries located in East Asia, Southeast Asia, Middle East, Africa and Europe infected cases of human infection with H5N1 virus were confirmed by laboratory diagnose, the total number of infected people is 288 and 170 of them died [Organization, W.H., Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1) Reported to WHO. 2007]. The epidemic spreading tendency did not cease, raising the entire world's attention.
Combining with the spread of H5N1 in recent years, its outbreak in poultry, its transmission to various mammals, its infection of human and its possible spread in mammals (Thanawongnuwech, R. et al., Emerg Infect Dis, 2005, 11 (5):699-701; Ungchusak, K. et al., N Engl J Med, 2005, 352 (4):333-40), large-scale outbreak of H5N1 viruses in humans is only a matter of time (Hien, T. T. et al., N Engl J Med, 2004, 351 (23):2363-5; Stohr, K. et al., N Engl J Med, 2005, 352 (4):405-7).
The first line of preventing the flu is neutralizing antibody, and the most effective antibody is produced by HA, so influenza H5N1 vaccine can be developed based on HA protein. However, the vaccine need to constantly “upgrade” in order to catch up with the pace of virus mutation, because the HA gene has high variability and antigenic drift can make the vaccine ineffective. In order to overcome it, each year, WHO needs to select new vaccine strains as vaccines for next year's epidemic season according to the monitoring result of the mutation of previous year's epidemic virus strains, and new vaccine needs to be injected each year to ensure the maintenance of effective protection against current epidemic virus strain. Large amount of traditional influenza vaccines were produced by chicken embryo culture method or cell culture method, they mainly are inactivated whole-virus vaccines, lytic vaccines or subunit vaccines. These traditional influenza vaccines continue to occupy the current market, but their immunogenicity, safety, and side effects are still not credible. Because H5N1 viruses have high toxicity to the chicken embryo, the original limited production capacity of vaccine manufacturers can't meet the requirements of the market at the global outbreak state. And the application of different kinds of live attenuated influenza vaccines was limited because of safety problems and side effects (Horimoto T. et al., Trends in Molecular medicine, 2006, 12(11):506-514). With the development of science and technology, using the new molecular biology technique to study new influenza vaccines has become a new trend of the present research of influenza vaccines, such as nucleic acid vaccine, genetically engineered vaccine, epitope vaccine.
At present, after long-term usage, two kinds of drugs have been confirmed to be able to treat influenza effectively, they are divided into two types: M2 ion channel inhibitors such as amantadine (amantadine) and rimantadine (rimantadine), and neuraminidase inhibitors such as oseltamivir (oseltamivir) and zanamivir (zanamivir) (Monto, A S Vaccine, 2003. 21 (16): 1796-800). The first two ones showed their anti-viral effects by suppressing the virus M2 ion channel protein, and the latter two can selectively inhibit the activity of neuraminidase on the virus surface, prevent the replication and release of progeny virus in host cells, prevent cold and alleviate the symptoms effectively. So, neuraminidase inhibitors are the relatively effective anti-influenza drugs. However, resistance to neuraminidase inhibitors in H5N1 have been continually discovered. And if taken within the first 2 days, it can shorten the course of diseases, even save the life of the patient. Although ion-channel inhibitors are very useful for some subtypes of influenza viruses (Dolin, R. et al., N Engl J Med, 1982. 307(10):580-4), it also causes serious side-effect, and thus, resistant strains appear soon (Shiraishi, K. et al., J Infect Dis, 2003, 188(1):57-61). And the resistant strains haven't shown weakened transmitting ability and pathogenicity. By now, resistance of ion-channel inhibitors appeared broadly over the world (Bright, R. A. et al., Lancet, 2005, 366(9492):1175-81), so ion-channel inhibitors were opposed for being used as therapeutic and preventive drugs against influenza by CDC of the United State in the 2005-2006 influenza season. However, considering many advantage of ion-channel inhibitors, it should be used for further treatment, but in combination with other drugs. The dosage shall be reduced as well as side-effect, and meanwhile, the risk of drug resistance shall be reduced (Tsiodras, S. et al., Bmj, 2007, 334 (7588):293-4). In 2005, Oseltamivir-resistant H5N1 virus strain was reported (Le, Q. M. et al., Nature, 2005, 437:1108; de Jong, M. D. et al., Engl J Med., 2005, 353:2667-2672).
Some infected and recovered patients of H5N1 have antibodies able to neutralize viruses in iv-vitro assay, which indicates that antibody may be one of the methods used for treating influenza virus infection (de Jong, M. D. et al., Nat. Med., 2006, 12: 1203-1207). Clinically, polyclonal and monoclonal antibodies are effectively used for preventing HAV, HBV, rabies and RSV infection (Sawyer, L. A., Antiviral Res., 2000, 47:57-77). During the 1918 spa-influenza, mortality was reduced by 50% by treatment using convalescent serum of human (Luke, T. C. et al., Ann Intern Med., 2006, 145:599-609). In China, the successful treatment experience of SARS and H5N1 patients also show that, patient convalescent serum could inhibit viral in-vivo replication effectively, so that dying patients could recover. In mouse models, H5N1 specific humanized mouse monoclonal antibodies, entirely humanized monoclonal antibodies and F(ab′)2 fragment have been proved to have efficacy for the prevention and treatment of H5N1 infection (Lu, J. et al., Respir Res., 2006, 7:43; Simmons, C. P. et al., PLOS Medicine, 2007, 4(5):928-936; Hanson, B. J. et al., Respir Res., 2006, 7:126). The persistent antigen mutation of highly pathogenic avian influenza H5N1 at receptor-binding site, namely antigenic drift, challenges the broad-spectrum anti-virus treatment ability of these antibodies. Thus, obtaining monoclonal antibodies with broad-spectrum neutralization ability becomes the hope for H5N1 treatment, and the use of high quality anti-H5N1 broad-spectrum neutralization monoclonal antibodies to treat H5N1 patients may inhibit in vivo replication of H5N1 virus and obtain ideal treatment effects. This is a brand new way of anti-virus treatment.
Furthermore, the study of molecular epidemiology shows that, about 30% of positively infected ducks do not have any symptom, 10% of the epidemic virus carrying chickens show no symptom. These infected animals can continually cause new infections for human, which causes huge threat to human health. All the related experts regard that, the spread of the H5 type highly pathogenic avian influenza virus in the entire eastern Asia, southeastern Asia and Europe shall be well controlled. Early phase diagnosis is the precondition, and then early quarantine, early handling can be done and human should be treated early.
It takes 4-5 days to diagnose avian influenza virus with traditional viral separation and serum diagnosis method, besides, the majority human and animal disease control system labs lack 3rd level biosafety lab, so the diagnose of H5 outbreak in southeastern Asia countries and regions is apparently lagging. The common situation is that there is still no diagnose report from labs after the death of large number of chickens and the completion of slaughtering, which causes great inconvenience for the control of virus outbreak. In addition, because a minority of poultry (especially waterfowl, such as domestic ducks) reveals no symptom of virus infection, and there is no effective testing means in the quarantine system, the above situation continues to develop, giving rise to repetitive outbreak of the virus in many countries and regions.
Meanwhile, since H5 avian influenza virus (among which, Goose/Guangdong/1/96 is the representative strain) belongs to highly pathogenic virus, it is fatal for the presently commonly used animal models, and the antigenicity of hemagglutinin protein (HA) expressed by genetic engineering method can't be completely obtained. Many of the world's famous laboratories have successively attempted to prepare monoclonal antibody against the virus series but they all failed. At present the virus antigenicity analysis has to use the monoclonal antibody prepared with A/chicken/Pennsylvania/1370/83(H5N2) and A/chicken/Pennsylvania/8125/83 (H5N2), whose specificity and reactivity obviously cannot meet the requirements of diagnostic reagent.
Because of the above situation, at present, a method for convenient, fast, and real time diagnosis is urgently required. Thus patients of the first cross-species infected generation can be isolated and treated, preventing the virus infection among human, and the transmission chain can be interrupted before the virus has adapted to human beings, so that the threat of human-wide influenza of the virus can be fundamentally eliminated.
Within China, studies on the detection of subtype H5 avian influenza virus have been reported in the literature. Qin Aijian et al. from College of Animal Husbandry and Veterinary Medicine, Yangzhou University (Qin Aijian, Shao Hongxia, Qian Xian et al., Journal of Chinese Prevention Veterinary Medicine 2003, No. 3) prepared hemagglutinin specific monoclonal antibodies against avian influenza viruses subtype H5 and subtype H9, and it was confirmed that with these monoclonal antibodies, the corresponding avian influenza virus can be quickly detected within 24 hours by indirect immunofluorescence assay. The detection time for the highly pathogenic subtype H5 of avian influenza virus was shortened to 4 hours, which was achieved by the Beijing Office for Entry-Exit Inspection and Quarantine with a rapid fluorescent RT-PCR. It was clinically confirmed on 10 Dec. 2005. Guo Yuanji reviewed in his paper with the title “Present Situation of Human Avian Influenza Research” that micro-neutralization experiment or ELISA with high specificity was needed for detecting the antibody against virus strain of subtype H5 (Guo Yuanji, Chinese Journal of Experimental and Clinical Virology, 2004, No. 3). But there is no research report on the detection of subtype H5 by ELISA
There have been research reports abroad on detection of H5N1 antibody using ELISA. Rowe et al. reported that using recombinant hemagglutinin protein as antigen envelope to detect H5N1 antibody indirectly by ELISA, the sensitivity of ELISA is 80%, and specificity is 62% (Rowe T, et al., J Clin Microbiol. April 1999; 37 (4): 937-43). But this literature is not directed to monoclonal antibody specific for hemagglutinin HA gene of subtype H5N1. Zhou et al. (Zhou E M et al., Avian Dis. 1998, 42 (4): 757-61) and Shafer et al. (Shafer, A L et al., Avian Dis. 1998, 42 (1): 28-34) detected avian influenza virus anti-core-protein antibody using competitive ELISA, but the detection subjects were all antibodies for NP proteins of all the subtypes H1-H16 of type A avian influenza, and the subtype couldn't be determined Lu reported a method for detecting avian influenza virus (AIV) by Dot-ELISA based on monoclonal antibody., This method detected AIV antigen directly and its specificity is that it has no cross-reaction with other avian viruses (Lu H. Avian Dis. 2003 47 (2): 361-9). Although Sala et al. established ELISA based on monoclonal antibody specific for surface glycoprotein of subtype H7, but the subtype was H7, and the monoclonal antibody was specific to surface glycoprotein (Sala G, Cordioli P, Moreno-Martin et al., Avian Dis. 2003, 47 (3 Suppl): 1057-9), rather than specific to hemagglutinin HA gene of subtype H5.
It is regretful that most of the monoclonal antibodies used in current immunology diagnosis of avian influenza virus are against core protein (NP protein), thus used for detecting influenza virus of type A. But type A influenza virus actually includes subtypes H1-H16 with 16 subtypes in total, among which most subtypes have no pathogenicity or only low pathogenicity while only subtype H5 of avian influenza virus is the most harmful avian influenza virus with high pathogenicity. Thus the available technologies are far from meeting the demands of clinic detections.
The ultimate aim of the present invention is to overcome the defects of present immune detection, treatment and prevention of AIV, the monoclonal antibody used is against hemagglutinin (HA) protein of H5 subtype. In mouse models, this monoclonal antibody can effectively treat infections of many types of H5N1 virus mutation strains, it can also be used to specifically detect highly pathogenic H5 subtype avian influenza virus and for the production of vaccine and other therapeutic drug against H5N1.